DESCRIPTION: This application, submitted in response to RFA-DA-16-004, proposes to functionally validate genetic variants identified in a NIDA-funded R01 (DA17305; PI E. Nelson) in which GWAS analyses in a sample limited to opioid misusers tested the hypothesis that genetic polymorphisms influence progression from exposure to severe opioid dependence. The strongest association was for SNPs in cornichon family AMPA receptor auxiliary protein 3 (CNIH3). Confirmation analyses performed in two samples with non-dependent opioid misusers yielded a genome-wide significant meta-analytic p of <4.3E-9 for rs10799590, an intronic CNIH3 SNP associated with reduced risk [OR 0.64 (0.54-0.74)].The large effect accounts for between 1.2 and 5.8% of the phenotypic variance. Furthermore, the association of CNIH3 is consistent with studies of opioids in animal models highlighting the importance of glutamate signaling. This application's R21 phase proposes to generate transgenic mice into which human CNIH3 has been introduced (both the risk and protective haplotypes) using innovative adaptations of CRISPR technology. The R33 phase proposes to use these mice to test our overarching hypothesis that CNIH3 polymorphisms alter gene expression impacting AMPA signaling and behavioral response to opioids. We will use behavioral assays related to opioid dependence (conditioned place preference, extinction, and reinstatement; context-dependent sensitization), which previously connected AMPA signaling to opioids. We will determine gene expression in response to morphine, focusing on addiction-related brain regions in which AMPA receptor subunit composition changes have been reported, to determine whether significant differences are found between mice with the protective and risk-associated haplotypes. We will also examine potential routes by which haplotype-associated effects may be mediated. The R21 component of the project's specific aim is: AIM 1 To generate humanized Cnih3 mice carrying the identified protective and risk-associated haplotypes The specific aims of the R33 component of the project are: AIM 1 To examine phenotypic differences between groups of transgenic mice with the protective and risk- associated haplotypes (with wild type and Cnih3 knockout mice as controls) in the expression of morphine CPP, its extinction, and reinstatement and context-dependent sensitization (CDS) AIM 2 To determine whether the protective haplotype alters Cnih3 expression at baseline (pre-opioids) or in response to morphine in these behavioral paradigms AIM 3 To examine whether these haplotypes alter Cnih3 and related protein content in the post-synaptic density of brain areas associated with morphine-induced behavioral responses AIM 4 To examine evidence for epigenetic mediation of altered gene expression associated with these SNPs in response to morphine CPP, including identifying SNPs that alter the activity of enhancers